Vol. 11, No. 2

MOLECULAR AND CELLULAR BIOLOGY, Feb. 1991, p. 987-1001

0270-7306/91/020987-15$02.00/0 Copyright © 1991, American Society for Microbiology

Spkl, a New Kinase from Saccharomyces cerevisiae, Phosphorylates Proteins on Serine, Threonine, and Tyrosine DAVID F. STERN,'* PAN ZHENG,2 DAVID R. BEIDLER,3 AND CYNTHIA ZERILLO' Departments of Pathology1 and Pharmacology3 and Immunobiology Section,2 Yale University School of Medicine, 310 Cedar Street, New Haven, Connecticut 06510 Received 26 June 1990/Accepted 16 November 1990

A Saccharomyces cerevisiae Agtll library was screened with antiphosphotyrosine antibodies in an attempt to identify a gene encoding a tyrosine kinase. A subclone derived from one positive phage was sequenced and found to contain an 821-amino-acid open reading frame that encodes a protein with homology to protein kinases. We tested the activity of the putative kinase by constructing a vector encoding a glutathione-Stransferase fusion protein containing most of the predicted polypeptide. The fusion protein phosphorylated endogenous substrates and enolase primarily on seine and threonine. The gene was designated SPKI for serine-protein kinase. Expression of the Spkl fusion protein in bacteria stimulated serine, threonine, and tyrosine phosphorylation of bacterial proteins. These results, combined with the antiphosphotyrosine immunoreactivity induced by the kinase, indicate that Spkl is capable of phosphorylating tyrosine as well as phosphorylating serine and threonine. In in vitro assays, the fusion protein kinase phosphorylated the synthetic substrate poly(Glu/Tyr) on tyrosine, but the activity was weak compared with serine and threonine phosphorylation of other substrates. To determine if other serine/threonine kinases would phosphorylate poly(Glu/Tyr), we tested calcium/calmodulin-dependent protein kinase II and the catalytic subunit of cyclic AMP-dependent protein kinase. The two kinases had similar tyrosine-phosphorylating activities. These results establish that the functional difference between serine/threonine- and tyrosine-protein kinases is not absolute and suggest that there may be physiological circumstances in which tyrosine phosphorylation is mediated by serine/threonine kinases.

Tyrosine-protein kinases are important regulators of cell growth and differentiation. These kinases were originally discovered as transforming proteins carried by oncogenic retroviruses and are now known to include receptors for many peptide growth factors. The cascade of events linking activation of Tyr kinases at the plasma membrane to cell division is not well understood, in part because of the difficulty in identifying physiologically relevant substrates. Previous efforts to identify Tyr kinase substrates have been based on biochemical approaches. As a complementary approach, it would be advantageous to identify Tyr kinases in systems in which genetic techniques could be used to identify genes encoding interacting proteins. For this reason we have endeavored to identify a Tyr kinase gene in -the budding yeast Saccharomyces cerevisiae. An advantage of yeast systems for studying Tyr kinases is that the yeast cell cycle is well characterized and in many regards resembles the vertebrate cell cycle. Some components of the cell cycle regulatory apparatus are highly conserved from yeasts to mammals, to the extent that mammalian homologs of yeast cell cycle-regulatory proteins can complement yeast cell cycle mutations (14, 40) and that important cell cycle proteins discovered in yeasts are regulated in the mammalian cell cycle (4, 23; reviewed in references 17 and 22). The use of nucleotide probes to identify yeast Tyr kinase genes has not been successful (16). However, extracts of budding yeast cells contain a phosphotransferase activity that phosphorylates a synthetic substrate on Tyr in vitro (26). Since nucleic acid homologies have not been useful in

*

the identification of yeast Tyr kinases, we have chosen to use a functional screen. Antibodies that recognize phosphotyrosine were used to screen a S. cerevisiae Agtll library. Immunoreactivity would be found if the recombinant phage encoded an enzyme capable of phosphorylating itself or bacterial proteins in the plaques (37). Antiphosphotyrosine antibodies have been used in immunoscreens to clone mammalian Tyr kinases (12). MATERIALS AND METHODS Antibody screen. Polyclonal rabbit antiphosphotyrosineGly-Ala-keyhole limpet hemocyanin was a gift from M. Kamps and B. M. Sefton (Salk Institute) or was prepared and affinity purified on a phosphotyramine column in this laboratory according to procedures explained elsewhere (11). A yeast genomic library (a gift from M. Snyder, Yale University) was produced by shearing S. cerevisiae S288C DNA, adding EcoRI linkers, and inserting it into Xgtll (30). Plaques (2 x 106) were screened with antiphosphotyrosine as described elsewhere (30) but with the filters blocked and hybridized according to protocols for antiphosphotyrosine immunoblotting (11). One phage, designated 9-1-2, induced strong immunoreactivity in plaques. Subclones. Bluescript SK9-15 was produced by subcloning a novel 3.6-kb EcoRI fragment from 9-1-2 into the EcoRI site of Bluescript SK (Stratagene Cloning Systems). SK15-9 was produced by recloning the insert from SK9-15 into Bluescript SK in the opposite orientation. The glutathione-Stransferase fusion plasmid GEX6-2b was produced by a three-part ligation of two contiguous fragments from SK15-9 (PvuII-182-BamHI-1383 and BamHI-1383-EcoRI-2733; numbering according to Fig. 2) with Glutagene GEX-1N

Corresponding author. 987

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(Amrad Corp., Ltd.) linearized with SmaI and EcoRI. For most experiments the negative control plasmid used was GEX-2T, which differs from GEX-1N by 33 bp (11 amino acids) at the polylinker, but GEX-IN did not induce antiphosphotyrosine immunoreactivity in bacterial lysates. Sequence. The nucleotide sequence of the insert in SK9-15 and SK15-9 was determined by producing random unidirectional deletions in the plasmids with exonuclease III and mung bean nuclease (Stratagene Cloning Systems) and then using the chain termination method for DNA sequencing with Sequenase (U.S. Biochemicals). The sequence shown in Fig. 2 is a composite of the sequences in both strands derived from SK9-15 and SK15-9. Protein and nucleic acid searches of National Biomedical Research Foundation (NBRF), EMBL, and GenBank databases and a collection of protein kinase sequences (kindly provided by Steve Hanks, Salk Institute) were conducted by using the WordSearch program from the University of Wisconsin Genetics Computer Group. Kinase assays. Overnight cultures of Escherichia coli HB101 carrying GEX6-2b or GEX-2T were diluted 1:10 into Luria broth supplemented with 50 ,ug of ampicillin per ml and incubated for 3 h at 37°C with shaking and then for 1 h with 1 mM IPTG (isopropyl thiogalactopyra.noside). GFX 2T and GEX6-2b cultures had similar growth properties, but for some experiments, GEX-2T and GEX6-2b culture densities were normalized by optical density (600 nm) before and after incubation with IPTG. All remaining operations were conducted at 0 to 4°C. Bacteria were collected by centrifugation and suspended in cold lysis buffer (1% [vol/vol] Triton X-100, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 2 mM EDTA, 10 ,ug of leupeptin per ml, 0.198 trypsin inhibitor units of aprotinin per ml, 0.1 mM phenylmethylsulfonyl fluoride in calciumand magnesium-free Dulbecco's phosphate-buffered saline (PBS- 2 mlV25 ml of culture). The suspensions were disrupted by sonication twice for 3 s each time at half-maximal power with a microprobe-equipped sonicator (Branson Ultrasonics Corp.) and cleared by centrifugation at top speed in a Brinkmann microcentrifuge for 5 min. Portions of lysates were incubated with glutathione-agarose (Sigma Chemical Co.; 50 RI at 50% [wt/vol] in lysis buffer per 300 to 800 ,ul of lysate) for at least 30 min with rotation. Antiphosphotyrosine was added at 1 to 2 p.g/ml of lysate and incubated for at least 30 min, and immune complexes were collected by incubation with protein A-Sepharose (Pharmacia CL4B) with rotation for at least 30 min. Glutathione-agarose and antiphosphotyrosine affinity complexes were washed twice in 0.5 ml of TG-V04 (1% [wt/vol] Triton X-100, 10% [vol/vol] glycerol, 0.198 TIU of Aprotinin per ml in PBS- with freshly added 100 ,uM sodium orthovanadate) and twice with 0.5 ml of yeast kinase buffer (20 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid]-NaOH [pH 7.4], 10 mM MgCl2, 10 mM MnCl2) and suspended in 50 to 100 ,ul of yeast kinase buffer. Rabbit muscle enolase (Boehringer Mannheim; denatured in 40 mM acetic acid for 10 min at 37°C) or poly(Glu/Tyr) (4:1; molecular mass, 20 to 50 kDa; Sigma) was used at 10 to 40 p.g/100 ul of reaction mixture. For in vitro phosphorylation, the washed complexes were incubated with 5 to 50 ,uCi of [y-32P]ATP (Amersham; specific activity greater than 5,000 Ci/mmol) for 10 to 15 min at 30°C. (Qualitatively similar results were obtained in experiments in which the ATP was diluted to a final concentration of 10 ,uM.) Following a brief centrifugation, the reaction supernatants were transferred to separate tubes.

MOL. CELL. BIOL.

The pellets were suspended in electrophoresis sample buffer containing 6 M urea and incubated at 100°C for 2 min (see Fig. 4). For the experiments in Fig. 8 and 9, the pellets were washed once in cold RIPA-V04 (10 mM sodium phosphate buffer [pH 7.4], 1% Triton X-100, 0.1% sodium dodecyl sulfate [SDS], 1% sodium deoxycholate, 150 mM NaCl, 0.198 trypsin inhibitor units of aprotinin per ml, 1 mM sodium orthovanadate), and once in PBS-. The supernatants were concentrated by the addition of nonlabeled ATP (to 23 mM), trichloroacetic acid (TCA; to 20%), and 100 ,ug of bovine gamma globulin carrier (omitted in some experiments). For samples phosphorylated without poly(GluITyr), equivalent amounts of poly(Glu/Tyr) were added to supernatants after the addition of TCA or to pellets after the addition of electrophoresis sample buffer to control for nonspecific or chemical association of 32p. TCA precipitates were incubated at 0°C for 1 to 2 h, collected by centrifugation in a microfuge at 4°C, and washed twice with cold 20% TCA, once with 95% ethanol, and once with ethanol-ether (50%

[vol/vol]).

Rat brain calcium/calmodulin-dependent protein kinase II and rabbit brain calmodulin (purified by DEAE chromatography, ammonium sulfate precipitation, Sephacryl S-400 chromatography, and binding to calmodulin; 6, 19) were gifts from Fred Gorelick (Yale Medical School). The kinase preparation contains no detectable cyclic AMP (cAMP)dependent, cGMP-dependent, or C kinase activity. For the experiment shown in Fig. 10, 500 ng of enzyme was added to 100 p.l of yeast kinase buffer containing 500 p.M CaCl2 and 2.5 p.g of bovine brain calmodulin. Incubations with [y_32p] ATP were as described above. Dilution of ATP to 10 p.M or shortening reaction times to 5 min did not alter results qualitatively. Reactions were terminated by the addition of ATP to 10 mM, EDTA to 20 mM, and TCA to 20%, and TCA precipitates were collected and washed as described above. The catalytic subunit of murine cAMP-dependent protein kinase purified from E. coli (28) was a gift from Wes Yonemoto and Susan Taylor (University of California, San Diego). Reactions were as just described except that CaCl2 and calmodulin were omitted and 350 ng of enzyme was used. Electrophoresis and phosphoamino acid analysis. Samples were analyzed by gel electrophoresis in 10 to 0.13% or 15 to 0.09% acrylamide-bisacrylamide SDS-polyacrylamide gels (27) or in nondenaturing gels prepared as described elsewhere (26), except that no detergents were used in the sample buffer or gels. After being run, nondenaturing gels were fixed without stain and then washed several times with 10% (vol/vol) acetic acid to rinse out residual ATP before a final rinse in water and drying for autoradiography. Phosphoamino acid analysis of gel-purified proteins and total reaction products have been described previously (3, 31). Immunoblotting with antiphosphotyrosine was performed as described elsewhere (11, 32). Nucleotide sequence accession number. The GenBank accession number for the sequence of SPKJ is M55623. RESULTS Identification of putative kinase gene. A genomic S. cerevisiae Xgtll library was screened with an affinity-purified antiphosphotyrosine antiserum. Phage 9-1-2 was characterized further because it induced the strongest immunoreactive signal. Infection of bacteria with phage 9-1-2 specifically induced the appearance of many bands in antiphosphotyrosine immunoblots (Fig. 1A; compare phage 9 with control

S. CEREVISIAE KINASE

VOL . 1 l, 1991

A. phage Xn

OD cD

B.

Bluescript C. Gex6-2B

a LO 0c) ur U:6,

m

cn

-

+

IPTG

200 200

200

A 4

97 97

9

68

97

68

IT

68 F: 43

43

43

43

:,..-j~.4-

FIG. 1. Antiphosphotyrosine immunoblot analysis of bacterial Cultures of E. coli Y1089 (1 ml each) were grown to an optical density at 600 nm of 0.4 and infected with three different phage from the Xgtll library that were designated 3-1 (phage 3), 9-1-2 (phage 9), and 10-1 (phage 10). Cultures were incubated overnight with IPTG (1 mM) except for the phage 9 culture on the left. (B) Cultures (1.0 ml) of bacteria carrying Bluescript SK9-15 or SK15-9 were grown to saturation. (C) An overnight culture of E. coli HB101 carrying plasmid GEX6-2b was diluted 1:10 into Luria broth containing 50 jig of ampicillin per ml and divided into equal portions that were incubated with shaking for 1 h at 37°C followed by 2 h in the absence (left) or presence (right) of 0.1 mM IPTG. Bacteria were harvested by centrifugation, frozen on dry ice, and thawed into electrophoresis sample buffer containing 6 M urea. Samples were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting with antiphosphotyrosine. Autoradiographs were exposed overnight (A) or for 3 days (C) to flashed film (Kodak X-Omat AR) with an intensifying screen at -70°C. Approximate molecular weights of prestained protein standards (BRL) are indicated. Antiphosphotyrosine immunoblots are of bacteria infected with phage (A) or carrying Bluescript subclones (B) or the glutathione-Stransferase fusion construct (C). lysates. (A)

phage 3 and 10). The presence of multiple immunoreactive bands was consistent with the idea that the phage induces a protein modification such as phosphorylation that affects many bacterial proteins (37) rather than simply encoding a single reactive species. 9-1-2 genomic DNA contained three EcoRI restriction fragments (0.7, 3.0, and 3.6 kb) not found in Xgtll, suggesting that this phage contains as much as 7.3 kb of inserted sequences. The three fragments were subcloned individually into Bluescript SK. The subcloned 3.6-kb fragment was biologically active: lysates of bacteria carrying this plasmid (denoted SK9-15) contain proteins that react with antiphosphotyrosine antibodies (Fig. 1B). Activity was also induced by the same insert cloned in the opposite orientation in SK (plasmid SK15-9), suggesting that cis regulatory sequences responsible for transcription in bacteria are present within the insert (Fig. 1B). Southern blotting experiments using the insert as probe showed that the sequences hybridize with DNA from S. cerevisiae, indicating that the inserted DNA

989

originated in yeast cells and not in lambda or a contaminating plasmid (1). Nucleotide and amino acid sequences. The insert consists of 3,663 bp counted from the beginning of the EcoRI site. A portion of the sequence is shown in Fig. 2, with the nucleotide numbered 1 located 929 bp from the beginning of the EcoRI site. A continuous open reading frame was found beginning at bp 908 from the EcoRI site (not shown in Fig. 2). The first Met codon in this open reading frame was the 19th codon and fits the Kozak consensus rules (13) for translational initiation. Since splicing occurs infrequently within coding sequences in S. cerevisiae, we have assumed that this Met is the initiator codon. The predicted polypeptide consists of 821 amino acids followed by a single termination site within the subclone. Kyte-Doolittle hydropathy analysis did not reveal any extended hydropathic regions that suggest a membrane-spanning domain. Although database searches did not reveal identity with other genes, the predicted polypeptide contains sequences spanning amino acids 1% to 469 that are characteristic of protein kinases. The consensus nucleotidc-binding Gly-X-Gly-X-X-(Gly) sequence begins at amino acid 205 and includes an unusual Ala rather than Gly at the final position. This and other regions of homology among protein kin.ies are depicted in Fig. 2 according to the system of Hanks et al.

(8). Two sequence domains (VI and VIII) distinguish known Ser/Thr kinases from Tyr kinases (8). According to these homologies, the presence of the amino acids Lys-311-ProAsp (region VI) and Gly-357-Thr-Leu-Ala-Tyr- Val-(AlaPro-Glu) (region VIII) predicts that the protein is a Ser/Thr kinase. To determine the relationship of this protein to other kinases, a comprehensive protein kinase sequence database consisting of kinase domain sequences only (obtained from S. Hanks) was searched for homologs by using the WordSearch program. The three most homologous kinases were cGMP-dependent protein kinase, myosin light-chain kinase (smooth-muscle form), and calcium/calmodulin-dependent protein kinase II. Searches of polypeptide sequences in the NBRF protein database (using the WordSearch program) also yielded cGMP-dependent protein kinase as the most homologous protein. A gapped alignment of the protein with bovine cGMPdependent protein kinase (34) is shown in Fig. 3. Most of the homologies lie within the kinase domain. Significantly, amino acids that are conserved in cyclic-nucleotide-binding sites (underlined in Fig. 3) do not appear to be present in our protein, suggesting that it is not likely to be a cyclicnucleotide-binding enzyme. We were also unable to identify a good consensus calmodulin-binding site (9, 15. 18), although the variability in primary sequence of calmodulinbinding sites may have obscured such a site. Database searches using only the amino- or carboxyl-terminal portions of the protein excluding the kinase domain and nucleotide searches of GenBank, EMBL, and NBRF databases failed to reveal any striking homologies, so the function of the nonkinase portions of the protein is uncertain. Activity of the kinase. The homology to vertebrate Ser/Thr kinases rather than Tyr kinases suggested that the protein is a Ser/Thr kinase. Such a kinase could have been cloned through cross-reactivity of the antiserum used for the immunoscreen. A second possibility was that the consensus rules derived by comparing kinases of multicellular eucaryotes do not hold for lower eucaryotes. The high degree of sequence homology between the two kinase families (8) suggests a

STERN ET AL.

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MOL. CELL. BIOL.

1 AG1A2GATAGTGTTACACAACATC0CTAAATGGAAATATTACACAAC 120 AGCATCCACGCAGGCTACTcAAAGGTTTTTGATTGAGAAGTTTTCTCAAGACAGATCGGC 29 1 N E N I T Q P T Q Q S T Q A T Q R F L I E K F S Q E Q I G

121 G2CATTGTGTGCAGGGTCATTTGTACCACGGGTCATTCCCATCCGAGATTTGTCAGCTGATATTTCACAGTGCTTAGGCGATCCATAAGAAGTTTGGACATTTGGT240 69 30 E N I V C R V I C T T G Q I P I R D L S A D I S Q V L K E K R S I K K V U T F G

6-2b fusion 1241 AG3AACCCAGCCTGTGACTATCATTTAGG0CATTTCAGACTGTCAAAGCATTTCCAAATACTACTAGGAGACGGTAACCTTTTATTGAAGACATTTCCACTAATGGGACC 360 109 70 R N P A C D Y H L G N I S R L S N K H F Q I L L G E D G N L L L N D I S T N G T

361 TGGTT4AATGGGC40AGTCGAGAGACAGCAATCAGTTACTGTCTCAGGTGATGAAATACCGTTGGTGTAGGCGTGGAATCAGATATTTTATCTCTGGTCATTTTCATCGAC 480 149 110 W L N G Q K V E K N S N Q L L S Q G D E I T V G V G V E S D I L S L V I F I N D

481 6A0TT0GCAGTGCCTCGAGCAGAACAAGTTGATCGCATAAGATCTACCTGAATACCTCTAATAGCTTCTCCTGGTCTTACATCATCTACTGCATCATCATGGTGGCCAC 600 189 150 K F K Q C L E Q N K V D R I R S N L K N T S K I A S P G L T S S T A S S N V A N

601 72GACTGGTA0TTTTGGATTTTTCGATTATTGACGAGTGGTGGGCCAGGGTGCATTTGCCACAGTAAGAAGCCATTGAAGACTACTGGGAAACATTCGCGGTGAAGATTATA n0 229 190 K T G I F K D F S I I D E V V G Q G A F A T V K K A I E R T T G K T F A V K I I * * kinase i* II

721 AGTAAACGCAAGTATAGGCATATGGATGGTGTGACAGAGAGTTAGAAGTATTGCAAGCTCAATCATCCAAGGATAGTACGATTGAAGGATTTTATGAAGATACTGAGAGTTAT 230 S K R K V I G N N D G V T R E L E V L Q K L N H P R I V R L K G F Y E D T E S Y IV III

840 269

841 TATATGGTGATGGAGTTCGTTTCTGGTGGTGACTTA9TGGATTTTGTTGCTGCTCATGGTGCGGTTGGAGAAGATGCTGGGAGGGAGATATCCAGGCAGATACTCACAGCAATAAATAC960 309 270 Y N V N E F V S G G D L M D F V A A H G A V G E D A G R E I S R Q I L T A I K Y V 961 310

1080 ATTCACTCTATGGGCATCAGCCATCGTGACCT18GCCCGAT0TATTCTTATTGACAGACGATCCTGTATTGGTAAGATACCGACTTTGGTCTGGCAAGTACAAGGAATGGG IH S

NG I S H R D L K P D N I L I E VI S S S

QD D P V L V K L T D F G L A K V a G N G VII

349

1081 TCTTTTATGA1ACCTTCTGTGGCACTTTGGCATATGTGGCACCTGAGTCATCAGAGGTGATACATCCGTATCTCCTGATGAATACGAAGAAGGATGAGTACTCTTCGTTAGTG1200 389 350 S F N K T F C G T L A Y V A P E V I R G K D T S V S P D E Y E E R N E Y S S L V S VIIIS 1201 GATATGTGGTC1TGGGATGTCTTGTGTATGTTATCCTACGGGCCACTTACCTTTTAGTGGTAGCACACAGGACCATTATATAACAGATTGGAGAGGCTCATATCATGAAGGGCCC1320 429 390 D N U S N G C L V Y V I L T G H L P F S G S T Q D Q L Y K a I G R G S Y H E G P IX X 1321 CTCA1GATTTCCGGATATCTGAGAAGCAAGAGATTTCATAGATTCATTGTTACAGGTGGATCCAAATATAGGTCGACAGCTGCAAAGCCTTGAATCATCCCTGGATCAAGATGAGT1440 469 430 L K D F R I S E E A R D F I D S L L 0 V D P N N R S T A A K A L N H P U I K N S 4- kinase XI 1441 CCATTGGGCTCACAATCATATGGTGATT1TTCACAATATCCTTATCACATCGTTGTCGCAGCAGAAATTATTAGAAATATGGACGATGCTCAATACGAATTTGTCAAGCGCAAGG1560 509 470 P L G S Q S Y G D F S 0 I S L S 0 S L S Q 0 K L L E N N D D A Q Y E F V K A 0 R

1561 TTCAGGATTTTACCCGCACACGCCCCTATTCGATATACACAGCCCGCATTGAA 1680 18ATTACAAATGGAGC0CAACTTCAGAACAGGATCAGGAGACCAGATGGAA 549 510 K L 0 N E Q Q L Q E Q D Q E D Q D G K I Q G f K I P A H A P I R Y T 0 P K S I E

FIG. 2. Nucleotide sequence and predicted amino acid sequence (beginning with the probable initiator methionine) of yeast gene. The nucleotide designated 1 is at position 929 from the beginning of the EcoRI site in SK9-15. The approximate beginning and ending of the protein kinase domain as predicted from sequence homologies (8) and the beginning of sequences incorporated into GEX6-2b are shown. Roman numerals indicate kinase consensus sequence domains, with conserved and less highly conserved amino acids in protein kinases (8) indicated with double or single underlines. *, Consensus ATP-binding motif Gly-X-Gly-X-X-(Gly); $, residues that discriminate known Ser/Thr kinases from Tyr kinases.

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1681 GCAGAAACTAGAGAACAAACTTTTACATTCCAATAATACTGAGAATGTCAAGAGCTCAAGAAAGGGTAATGGTAGGTTTTTAACTTTAAACCATTGCCTGACAGCATTATTCAA 550 A E T R E Q K L L H S N N T E N V K S S K K K G N G R F L T L K P L P D S I I Q

1800 589

15801 GEEAGCCGGGAGATTCAGCVPGGTGTGCCATTTTTCATTGGTAGATCCGAGGATTGCTGTNTTGALGACTAGGTTGTCTCGAGTCATTGCTTCATTTTCFKGAGG 590 E S L E I 0 Q G V N P F F I G R S E D C N C K I E D N R L S R V H C F I F K K R

1920 629

1921 CATGCTGTAGGCAGCATGTATGATCCCGGCACAGGTTTAGATGATATTTGGTATTGCCACACCGGACTAACGTGAGCTATTTA T CCGCATGATACAGGGTACGA 630 H A V G K S N Y E S P A 0 G L D D I W Y C H T G T N V S Y L N N N R N I Q G T K

2040 669

2041 TTCCTTTTACAAGACGGAGATGAATCAAGATCATTTGGGATAAACAAAATTTGTCATTGGCTTTAAMGTGGAAATACGATACTACAGGTCTGTTTAACGAGGGATTAGGTATG 670 F L L 0 D G D E I K I I W D K N N K F V I G F K V E I N D T T G L F N E G L G N

2160 709

2161 TTACAAGAACAAGAGTAGTACTTAAGCAAACAGCCGAAGAAAAGATTTGGTGAAAAGTTAACCCAGATGATGGCAGCTCAACGTGCAAATCAACCCTCGGCTTCTTCTTCATCAATG 710 L 0 E 0 R V V L K 0 T A E E K D L V K K L T Q N N A A Q R A N Q P S A S S S S N

2280 749

TCGGCTAAGAAGCCGCCAGTTAGCGATAC'ATATAACGGCAATAATTCGGTACTAAACGACTTGGTA'GAGTCACCGA;TAATGCGAATACGGGGMCATTTTGAAGAGAATACATTCG

2281 750 S A K K P P V S D T N N N G N N S V L N D L V E S P I N A N T G N

I

L K R

I

H S

2401 GTAAGTTTATCGCAATCACAAATTGATCCTAGTAAGAAGGTTAAAGGGCAAAATGGACCAAACCTCAAGGCCCCGAGAATTTGCAATTTTCGTAACCAAGGACAAATACCCATAGA 790 V S L S Q S Q I D P S K K V K R A K L D Q T S K G P E N L Q F S

2521

AATGCTGCCCCTTTTTAAGAGAGAAGATGGTAGATACC'

MTACTCAGAA TTCCCAGTACAAAGAACCA

TATCGGA GTCAATAAC

CTTGCTTTCGCA TAAAGT

GTATGAGA

2641 ATCACTCAGAAGCACCCAGTAATAAAGGATGCAGATAGCTCGAGATTTGGTAAGGTTGAGTTTAGGGACTTTTATGACGAAGTTTCACGGATTC

2400 789

2520 821

2640

2735

FIG. 2-Continued.

common evolutionary origin, and a primordial Tyr kinase might resemble a Ser/Thr kinase. To determine the specificity of the putative kinase, we first analyzed extracts of bacteria carrying SK9-15 that were labeled metabolically with 32Pi. SK9-15 did not induce the production of phosphate-labeled bands that differed from those found in bacteria carrying a control plasmid (30a). Phosphoamino acid analysis of total lysates or size-fractionated proteins did not reveal any differences. Similar experiments performed on bacteria infected with phage 9-1-2 also failed to reveal any difference clearly attributable to the gene. However, because of the basal production of phosphotyrosine in these experiments (probably resulting from hydrolysis of nucleotidylated bacterial proteins; 5) we were not confident that a small increment in phosphotyrosine would be detected. In a second approach, we used the bacterial expression vector pGEX-1N to produce an in-frame fusion of the carboxyl terminus of glutathione-S-transferase (29) to amino acid 51 of the protein (Fig. 4A). The polypeptide encoded by plasmid GEX6-2b should consist of 28 kDa of glutathioneS-transferase fused to 771 amino acids of the yeast protein and have a molecular mass of approximately 105 kDa. Antiphosphotyrosine immunoblots of extracts from bacteria carrying GEX6-2b revealed many reactive bands (Fig. 1C, right, and Fig. 5D). No immunoreactive material was seen in bacteria carrying GEX-1N or the related vector GEX-2T only (Fig. SD) or GEX6-2b cultured without IPTG induction (Fig. 1C, left). To determine whether the reactivity was due to tyrosine phosphorylation or to cross-reactivity of the antibodies with other phosphoamino acids, we attempted

to block antibody binding with individual phosphoamino acids (Fig. 5). As a positive control we used lysates prepared from B104-1-1 cells, which are NIH 3T3 cells transformed with the neu oncogene, which encodes a bona fide tyrosine kinase (31, 33). We have previously shown that reactivity of these sera to the neu protein is due to tyrosine phosphorylation (32, 33). Lysates from neu-transformed cells and GEX6-2b-carrying bacteria contained many immunoreactive species, but lysates from bacteria harboring GEX-2T did not (Fig. SD). The immunoreactivity was only slightly affected by the presence of excess phosphoserine and phosphothreonine (Fig. SA and B) but was completely abolished by the presence of phosphotyrosine (Fig. SC). These data bolstered the conclusion that the immunoreactivity in GEX6-2b lysates derived from the presence of phosphotyrosine. Phosphorylation of bacterial proteins. Since the glutathione-agarose fusion protein system should permit high-level expression of the fusion protein, we now repeated the phosphate-labeling experiments that we had attempted earlier with the Bluescript subclones. Parallel cultures of GEX-2T and GEX6-2b bacteria were grown to similar densities, induced with IPTG, and labeled metabolically with 32p;. The bacterial proteins were resolved by electrophoresis and transferred to an Immobilon P membrane (Fig. 6A). Overall phosphate labeling of proteins was greatly enhanced in bacteria containing the GEX6-2b fusion gene (Fig. 6A), but staining with Coomassie brilliant blue showed that similar amounts of protein were present in the two lysates (not shown). A nonradioactive lysate from 6-2b bacteria was analyzed by antiphosphotyrosine immunoblotting on a neighboring lane of the same filter (not shown). The major

Y kin

G kin 51 Y

G Y G Y

.....HENITQPTQQSTQATQRFLIE 21

1

THIGPRTTRAQGISAEPQTYRSFHDLRQAFRKFTKSERSKDLIKEAILDN

100

22 KF..... SQEQIGENIVCRVICTTGQIPIRD ....... LSADISQVLKE 58

101

DFMKNLELSQIQEIVDCXYPVEYGXDSCIIKEGDVGSLVYVMED_KVEVT

. 59 KRSIK.......... .... .e.KVWTFGRN

.

150

PACD 75

151 KEGVKLCTMGPGKVFGEhLILYNCT TATVKTLVNVKLWAIDRQCFQTIX 200

.QILLGEDGNLLLNDISTNGTWLNGQ 114

76 YHLGNISRLSNKHF .......

XRTGLIKHTEYMEFLKSVPTFQSLPEEILSKLADVLEETHYENGEYIIRQ 250

G

201

Y

115 KVEKNSNQLLSQGD.EITVGVGVESDILSLVIFINDKF..KQCLEQNKV. 160

G

251 GARGDTZFIISKKGVNVTREDSPNEDPVFLRTLGKGDWFGEKALQGEDVR 300

Y

161

G

301 TANVIAAEAVTCLVIDRDSFKHLIGGLDDVSNKAYEDAEAKAKYEAEAAF 350

Y

194 FKDFSIID... .EVVGQGAFATVK.KAIERTTGKTFAVKIISKRKVIGNM 238

G

351 FANLKLSDFNIIDTLGVGGFGRVELVQLKSEESKTFAMKILKKRHIVDTR 400

Y

239 DG..VTRELEVLQKLNHPRIVRLKGFYEDTESYYMVMEFVSGGDLMDFVA 286

G

401 QQEHIRSEKQIMQGAHSDFIVRLYRTFKDSKYLYMLMEACLGGELWTILR 450

Y

287 AHGAVGEDAGREISRQILTAIKYIHSMGISHRDLKPDNILIEQDDPVLVK 336

G

451 DRGSFEDSTTRFYTACVVEAFAYLHSKGIIYRDLKPENLIL..DHRGYAK 498

Y

337 ITDFGLAKVQGNGSFMKTFCGTLAYVAPEVIRGKDTSVSPDEYEERNEYS 386

G

499 LVDFGFAKKIGFGKKTWTFCGTPEYVAPEIILNKGHDISA.. 538 . .......

Y

387 SLVDMWSMGCLVYVILTGHLPFSGSTQDQLYKQIGRGSYHEGPLKDF..R 434

G

539 ... DYWSLGILMYELLTGSPPFSGPDPMKTYNIILRGI.... DMIEFPKK 581

Y

435 ISEEARDFIDSLLQVDPNNRSTAAKALNHPWIKMSPLGSQSYGDFSQISL 484

G

582 IAKNAANLIKKLCRDNPSERLGNLKNGVKDIQKHKWFEGFNWEGLRKGTL 631

Y

485 SQSLSQQKLLENMDDAQYEFVKAQRKLQMEQQLQEQDQEDQDGKIQGFKI 534

G

632 TPPII.PSVASPTDTSNFD.........e. eSFPEDNDEPPPDDNSGWDI 668

Y

535 PAHAPIRYTQPKSIEAETREQKLLHSNNTENVKSSKKKGNGRFLTLK--'-(821)

G

669 DF..........................

DRIRSNLKNTSKIASPGLTSSTASSMVANKTGI 193

.

I-:: : . : :

* *

*

* 1-----1..

-.j111:11

..

***-

::.1 1:11-11 *11111:1::

I.I-*

I1 .11111 .11111:1 *1: *:I:

I 11:1-1:1 :111

III1-.- * I- I II

.:1

670

FIG. 3. Alignment of yeast polypeptide with bovine cGMP-dependent protein kinase. Polypeptides were aligned by using the University of Wisconsin Genetics Computing Group Gap program. The overall sequence identity was 25%, and similarity was 49%. The tandem cGMP-binding regions of cGMP kinase are indicated with boldface type, with arrows showing the junction of these two domains after amino acid 227. Amino acids conserved in cyclic-nucleotide-binding proteins are underlined (38). *, Position of the consensus ATP-binding site; Y kin, predicted yeast polypeptide; G kin, cGMP-dependent protein kinase. 992

S. CEREVISIAE KINASE

VOL. 11, 1991

993

A. EcoRl

EcoRl

Pvull

SK9-15 I

821

kinase

glutathione-S-transferase

B. kinase assays at-PDEIIp 0g

m N

m

N

Gex6-2B

I

f